RK-286C

We have established a bioassay system using a mouse cdc2 mutant cell line, tsFT210, to detect inhibitors of the mammalian cell cycle. When cultured at the high temperature, restrictive temperature at 39.4 degrees C, tsFT210 cells can be arrested at G2 phase and are large in size. Four hours after release from G2 arrest, the cells entered into the G1 phase. At this time, G1 phase cells were easily discriminated from the G2/M-cells by their size under microscopic observation. The cell-morphology-based bioassay utilizing tsFT210 cells is very simple and sensitive for detecting cdc2 kinase inhibitors and also G2/M-phase inhibitors of the mammalian cell cycle. To demonstrate the merits of this bioassay, the effects of protein kinase inhibitors isolated from actinomycetes were investigated. RK-286C and RK-1409, which are structurally related to staurosporine, inhibited cell cycle progression at the G2 phase in both G2-synchronized and nonsynchronized cultures of tsFT210 cells. Another kinase inhibitor, sangivamycin, inhibited cell cycle progression at the G2 phase of cells released from temperature arrest but did not inhibit that of the exponentially growing cells. Using the bioassay system, we carried out screening of the cell cycle inhibitors from the microbial metabolites and have discovered several new inhibitors, including novel compounds such as tryprostatins A, B and acetophthalidin. Thus, this bioassay allowed for the detection of cell cycle inhibitors and provided a convenient and useful method for the screening of new inhibitors from the microbial metabolites.

A novel inhibitor of protein kinase C was found in the fermentation of a soil actinomycete, strain RK-1409. According to the taxonomic studies, the producing strain was designated as Streptomyces platensis subsp. malvinus RK-1409. The protein kinase C inhibitor, RK-1409 (7-oxostaurosporine) inhibited the morphological change of a human chronic erythroleukemia cell, K-562, induced by phorbol 12,13-dibutyrate (PDBu) at the concentration of 10 ng/ml. The concentration of 3 ng/ml inhibited the activity of protein kinase C in vitro. RK-1409 inhibited the cell cycle progression at G2 phase of K-562 cells.

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.